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Identification of a plastid intercistronic expression element (IEE) facilitating the expression of stable translatable monocistronic mRNAs from operons.

Zhou F, Karcher D, Bock R - Plant J. (2007)

Bottom Line: At least some polycistronic transcripts are not translatable, and endonucleolytic processing may therefore be a prerequisite for translation to occur.As the requirements for intercistronic mRNA processing into stable monocistronic transcript are not well understood, we have sought to define minimum sequence elements that trigger processing and thus are capable of generating stable translatable monocistronic mRNAs.We describe here the in vivo identification of a small intercistronic expression element that mediates intercistronic cleavage into stable monocistronic transcripts.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Molekulare Pflanzenphysiologie (MPI-MP), Am Mühlenberg 1, D-14476 Potsdam-Golm, Germany.

ABSTRACT
Most plastid genes are part of operons and expressed as polycistronic mRNAs. Many primary polycistronic transcripts undergo post-transcriptional processing in monocistronic or oligocistronic units. At least some polycistronic transcripts are not translatable, and endonucleolytic processing may therefore be a prerequisite for translation to occur. As the requirements for intercistronic mRNA processing into stable monocistronic transcript are not well understood, we have sought to define minimum sequence elements that trigger processing and thus are capable of generating stable translatable monocistronic mRNAs. We describe here the in vivo identification of a small intercistronic expression element that mediates intercistronic cleavage into stable monocistronic transcripts. Separation of foreign genes by this element facilitates transgene stacking in operons, and thus will help to expand the range of applications of transplastomic technology.

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Related in: MedlinePlus

Foreign protein accumulation in transplastomic lines harboring various candidate processing elements between the nptII and yfp cistrons(a) Western blot detection of NptII and YFP protein accumulation in transplastomic lines. Total soluble proteins were separated by denaturing gel electrophoresis, blotted and probed with anti-NptII or anti-GFP antibodies. As a loading control, accumulation of the large subunit of Rubisco (RbcL) is shown. Whereas the NptII protein accumulates to comparable levels in all transplastomic lines, YFP accumulation is restricted to Nt-pZF75 lines (containing the ±25 IEE from the psbT–psbH intergenic spacer), correlating with accumulation of stable monocistronic yfp message only in these lines.(b) Detection of YFP fluorescence in Nt-pZF75 lines by confocal laser-scanning microscopy. Yellow YFP fluorescence, red fluorescence of the chlorophyll, and the overlay of the two fluorescences are shown for the wild-type and an Nt-pZF75 transplastomic line.(c) Comparison of YFP accumulation in Nt-pZF75 transplastomic lines with GFP accumulation in control transplastomic plants expressing GFP from the Prrn–Trps16 expression cassette (Nt-pDK60). The GFP signal in Nt-pDK60 plants is stronger than the YFP signal in Nt-pZF75 lines, which may be due to weaker recognition of YFP by the anti-GFP antibody and/or lower stability of YFP in plastids. A dilution series with purified GFP protein (rGFP) is also shown. Immunological detection of the Rubisco large subunit (RbcL) was performed as a loading control.
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fig05: Foreign protein accumulation in transplastomic lines harboring various candidate processing elements between the nptII and yfp cistrons(a) Western blot detection of NptII and YFP protein accumulation in transplastomic lines. Total soluble proteins were separated by denaturing gel electrophoresis, blotted and probed with anti-NptII or anti-GFP antibodies. As a loading control, accumulation of the large subunit of Rubisco (RbcL) is shown. Whereas the NptII protein accumulates to comparable levels in all transplastomic lines, YFP accumulation is restricted to Nt-pZF75 lines (containing the ±25 IEE from the psbT–psbH intergenic spacer), correlating with accumulation of stable monocistronic yfp message only in these lines.(b) Detection of YFP fluorescence in Nt-pZF75 lines by confocal laser-scanning microscopy. Yellow YFP fluorescence, red fluorescence of the chlorophyll, and the overlay of the two fluorescences are shown for the wild-type and an Nt-pZF75 transplastomic line.(c) Comparison of YFP accumulation in Nt-pZF75 transplastomic lines with GFP accumulation in control transplastomic plants expressing GFP from the Prrn–Trps16 expression cassette (Nt-pDK60). The GFP signal in Nt-pDK60 plants is stronger than the YFP signal in Nt-pZF75 lines, which may be due to weaker recognition of YFP by the anti-GFP antibody and/or lower stability of YFP in plastids. A dilution series with purified GFP protein (rGFP) is also shown. Immunological detection of the Rubisco large subunit (RbcL) was performed as a loading control.

Mentions: Having established that monocistronic nptII transcripts accumulate in all transplastomic lines, whereas stable monocistronic yfp mRNA accumulates only in the Nt-pZF75 lines, we next wished to investigate the correlation between RNA abundance and protein accumulation. The high-level kanamycin resistance of all transplastomic lines tentatively indicated that NptII protein accumulates to reasonably high levels (Figure 3, and data not shown). This was confirmed by Western blot analysis with a specific anti-NptII antibody: all lines accumulated similarly high levels of NptII protein (Figure 5a), as expected from the accumulation of similar amounts of monocistronic nptII message in all transplastomic lines (Figure 4a). In contrast, when the blots were probed with an anti-GFP antibody (which also recognizes YFP, because YFP is a mutant GFP variant), protein accumulation was only detected in the Nt-pZF75 lines, correlating with the accumulation of stable monocistronic mRNA only in these lines. We therefore conclude that the complete stem–loop structure (±25) surrounding the psbT–psbH intercistronic processing site (as present in the Nt-pZF75 lines) represents a suitable sequence element to confer stable expression of downstream cistrons in multi-gene operons, and thus can serve as a genuine IEE.


Identification of a plastid intercistronic expression element (IEE) facilitating the expression of stable translatable monocistronic mRNAs from operons.

Zhou F, Karcher D, Bock R - Plant J. (2007)

Foreign protein accumulation in transplastomic lines harboring various candidate processing elements between the nptII and yfp cistrons(a) Western blot detection of NptII and YFP protein accumulation in transplastomic lines. Total soluble proteins were separated by denaturing gel electrophoresis, blotted and probed with anti-NptII or anti-GFP antibodies. As a loading control, accumulation of the large subunit of Rubisco (RbcL) is shown. Whereas the NptII protein accumulates to comparable levels in all transplastomic lines, YFP accumulation is restricted to Nt-pZF75 lines (containing the ±25 IEE from the psbT–psbH intergenic spacer), correlating with accumulation of stable monocistronic yfp message only in these lines.(b) Detection of YFP fluorescence in Nt-pZF75 lines by confocal laser-scanning microscopy. Yellow YFP fluorescence, red fluorescence of the chlorophyll, and the overlay of the two fluorescences are shown for the wild-type and an Nt-pZF75 transplastomic line.(c) Comparison of YFP accumulation in Nt-pZF75 transplastomic lines with GFP accumulation in control transplastomic plants expressing GFP from the Prrn–Trps16 expression cassette (Nt-pDK60). The GFP signal in Nt-pDK60 plants is stronger than the YFP signal in Nt-pZF75 lines, which may be due to weaker recognition of YFP by the anti-GFP antibody and/or lower stability of YFP in plastids. A dilution series with purified GFP protein (rGFP) is also shown. Immunological detection of the Rubisco large subunit (RbcL) was performed as a loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230500&req=5

fig05: Foreign protein accumulation in transplastomic lines harboring various candidate processing elements between the nptII and yfp cistrons(a) Western blot detection of NptII and YFP protein accumulation in transplastomic lines. Total soluble proteins were separated by denaturing gel electrophoresis, blotted and probed with anti-NptII or anti-GFP antibodies. As a loading control, accumulation of the large subunit of Rubisco (RbcL) is shown. Whereas the NptII protein accumulates to comparable levels in all transplastomic lines, YFP accumulation is restricted to Nt-pZF75 lines (containing the ±25 IEE from the psbT–psbH intergenic spacer), correlating with accumulation of stable monocistronic yfp message only in these lines.(b) Detection of YFP fluorescence in Nt-pZF75 lines by confocal laser-scanning microscopy. Yellow YFP fluorescence, red fluorescence of the chlorophyll, and the overlay of the two fluorescences are shown for the wild-type and an Nt-pZF75 transplastomic line.(c) Comparison of YFP accumulation in Nt-pZF75 transplastomic lines with GFP accumulation in control transplastomic plants expressing GFP from the Prrn–Trps16 expression cassette (Nt-pDK60). The GFP signal in Nt-pDK60 plants is stronger than the YFP signal in Nt-pZF75 lines, which may be due to weaker recognition of YFP by the anti-GFP antibody and/or lower stability of YFP in plastids. A dilution series with purified GFP protein (rGFP) is also shown. Immunological detection of the Rubisco large subunit (RbcL) was performed as a loading control.
Mentions: Having established that monocistronic nptII transcripts accumulate in all transplastomic lines, whereas stable monocistronic yfp mRNA accumulates only in the Nt-pZF75 lines, we next wished to investigate the correlation between RNA abundance and protein accumulation. The high-level kanamycin resistance of all transplastomic lines tentatively indicated that NptII protein accumulates to reasonably high levels (Figure 3, and data not shown). This was confirmed by Western blot analysis with a specific anti-NptII antibody: all lines accumulated similarly high levels of NptII protein (Figure 5a), as expected from the accumulation of similar amounts of monocistronic nptII message in all transplastomic lines (Figure 4a). In contrast, when the blots were probed with an anti-GFP antibody (which also recognizes YFP, because YFP is a mutant GFP variant), protein accumulation was only detected in the Nt-pZF75 lines, correlating with the accumulation of stable monocistronic mRNA only in these lines. We therefore conclude that the complete stem–loop structure (±25) surrounding the psbT–psbH intercistronic processing site (as present in the Nt-pZF75 lines) represents a suitable sequence element to confer stable expression of downstream cistrons in multi-gene operons, and thus can serve as a genuine IEE.

Bottom Line: At least some polycistronic transcripts are not translatable, and endonucleolytic processing may therefore be a prerequisite for translation to occur.As the requirements for intercistronic mRNA processing into stable monocistronic transcript are not well understood, we have sought to define minimum sequence elements that trigger processing and thus are capable of generating stable translatable monocistronic mRNAs.We describe here the in vivo identification of a small intercistronic expression element that mediates intercistronic cleavage into stable monocistronic transcripts.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Molekulare Pflanzenphysiologie (MPI-MP), Am Mühlenberg 1, D-14476 Potsdam-Golm, Germany.

ABSTRACT
Most plastid genes are part of operons and expressed as polycistronic mRNAs. Many primary polycistronic transcripts undergo post-transcriptional processing in monocistronic or oligocistronic units. At least some polycistronic transcripts are not translatable, and endonucleolytic processing may therefore be a prerequisite for translation to occur. As the requirements for intercistronic mRNA processing into stable monocistronic transcript are not well understood, we have sought to define minimum sequence elements that trigger processing and thus are capable of generating stable translatable monocistronic mRNAs. We describe here the in vivo identification of a small intercistronic expression element that mediates intercistronic cleavage into stable monocistronic transcripts. Separation of foreign genes by this element facilitates transgene stacking in operons, and thus will help to expand the range of applications of transplastomic technology.

Show MeSH
Related in: MedlinePlus